Methyl-branched fatty acids and their biosynthesis in the housefly, Musca domestica L. (Diptera: Muscidae)

Insect Biochem. Molec. Biol. Vol. 24, No. 8, pp. 803 810, 1994

Pergamon

0965-1748(94)E0006-3

Copyright (~:~1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0965-1748/94 $7.00 + 0.00

Methyl-branched Fatty Acids and their Biosynthesis in the Housefly, Musca domestica L. (Diptera:Muscidae) GARY J. BLOMQUIST,t~ LIN GUO,~f PEIDE GU,t CARIANNE BLOMQUIST,t RONALD C. REITZ,t JAMES R. REEDt Received 1 November 1993; revised and accepted 21 January 1994

Eighteen methyl-branched fatty acids from the housefly, Musca domestica L. (Diptera:Muscidae) were identified by gas chromatography-mass spectrometry after reduction to the corresponding hydrocarbons. A deuterium was inserted on what was the carboxyl carbon and the straight chain components were removed by molecular sieve. The deuterium allowed the mass spectral determination of the methyl branch position with respect to the carboxyl end of the parent fatty acid. The methyl-branched fatty acids were characterized as n-2, n-3, n-4, n-5, n-6, n-7, n-8 and n-9 monomethyl fatty acids of 15-19 carbons and a n-3,7 dimethyl fatty acid of 18 total carbons. These methyl-branched fatty acids have similar branching patterns and are presumed to be the precursors to the methyl-branched hydrocarbons, some of which function as an arrestant in the female sex pheromone. With increasing concentrations of methylmalonyl-CoA, its incorporation into methyl-branched fatty acids increased in both day 1 and day 4 males and females using both microsomal and soluble fatty acid synthases (FAS). Methylmalonyl-CoA inhibited both the soluble and microsomal FAS in a competitive manner. The data on the incorporation of methylmalonyl-CoA into methyl-branched fatty acids by day 1 and day 4 males and females indicate that the regulation of methyl-branched hydrocarbon synthesis does not reside at the level of fatty acid synthesis, but must occur during the process of the fatty acyl-CoA elongation or reductive conversion of very long chain fatty acyl-CoAs to hydrocarbons. Methyl-branched fatty acids Methyl-branched hydrocarbon Methylmalonyl-CoA

INTRODUCTION Long chain hydrocarbons are often major components of insect cuticular lipids, where they provide a barrier to prevent desiccation, and in some species, including the housefly Musca domestica L., serve in chemical communication (Howard and Blomquist, 1982; Howard, 1993; Blomquist et al., 1993). In the housefly, methylalkanes between C28 and C38 are part of the female sex pheromone (La-France et al., 1989) and some of the components serve as an arrestant during the mating process (Uebel et al., 1976; Adams and Holt, 1987; Adams, Nelson and Fatland, pers. commun.). 4-day old females have large amounts of methyl-branched hydrocarbons, whereas only very low amounts are present in newly emerged females and in males of all ages (Nelson et al., 1981; Blomquist et al., 1987a).

t D e p a r t m e n t of Biochemistry, University of Nevada, Reno, NV 89557-0014, U.S.A. SAuthor for correspondence. 803

Fatty acid synthetase Microsomal FAS

Radiotracer studies showed that female houseflies incorporate labeled acetate and propionate into methylbranched hydrocarbon (Dillwith et al., 1981). RadioGLC analysis showed that [1-14C]propionate labeled only methyl-branched alkanes, and thus, the incorporation of [1-'4C]propionate was used to monitor methylbranched hydrocarbon synthesis. Methyl-branched hydrocarbon synthesis increased dramatically in females 2 days after emergence, and reached the highest level at day 4 (Dillwith et al., 1983; Adams et al., 1984). In contrast, male houseflies synthesized only minimal amounts of methyl-branched hydrocarbons during the entire adult stage. However, when treated with 20hydroxyecdysone, male flies were induced to synthesize increased levels of methyl-branched hydrocarbons (Blomquist et al., 1984). The work reported herein was designed to determine if methyl-branched fatty acids with the same methyl branching pattern as the major methyl-branched hydrocarbons were present in the housefly, and to examine the effect of age and sex on the ability of soluble and

GARY J. BLOMQUIST et al.

804

microsomal FASs to incorporate methylmalonyl-CoA into fatty acids to determine if the production of methylbranched fatty acids was regulated at the level of FAS.

MATERIALS AND M E T H O D S

Houseflies Pupae of the Fales T-II strain houseflies, M. domestica, were provided by the Biology section, S. C. Johnson & Sons, Racine, Wis. Within 24 h after emergence, male and female insects were separated, and adult males and females were maintained separately and fed ad lib. on sucrose-low-fat powdered milk (1:1 w/w) and water at 2Y~C.

Products, DuPont Co., Boston, Mass. CoA derivatives, reduced nicotinamide adenine dinucleotide phosphate ( N A D P H ) , dithioerythritol (DTE), ethylenediaminetetraacetic acid (EDTA), leupeptin and bovine serum albumin (BSA) were purchased from Sigma Chemical Company, St Louis, Mo. All solvents except diethyl ether were redistilled in glass prior to use.

Isolation of microsomal and soluble .fractions All procedures were performed on ice or at 4~C. Whole houseflies were homogenized in a homogenization buffer (0.05M potassium phosphate buffer

16:0 16:1 /

Characterization of methyl-branched fatty acids Total lipid was extracted from day 4 female houseflies by the method of Bligh and Dyer (1959), and saponified at 8ff'C with 0.5 M K O H for 60 min. After acidification with HC1, lipid was extracted with chloroform, and the fatty acids were separated from other lipids by T L C in hexane:diethyl ether (80:20), and subsequently converted into fatty acid methyl esters with 14% BF 3methanol. The fatty acid methyl esters were reduced to the corresponding alcohols, converted to bromides and then to hydrocarbons by the methods described in Juarez et al. (1992), except that the conversion of the alkyl bromide to alkane used NaBD4 rather than NaBH4. This produced a monodeuterated alkane with the deuterium present on the carbon that had been the carboxyl carbon. The saturated components of the hydrocarbon fraction were obtained by hexane elution through 20% AgNO3 on BioSil A (Bio-Rad Labs, Richmond, Calif.) mini columns, and the methylbranched components were separated from the straightchain components by inclusion of the latter in 5 A molecular sieves in isooctane (O'Connor et al., 1962). Gas chromatographic (GC) analyses of total fatty acids, their reduction products and the molecular sieve products were performed on a Hewlett Packard 5890 gas chromatograph which was controlled by a Hewlett Packard 3393A integrator G C terminal. Samples were analyzed on a 30 m × 0.32 m m I.D., 0.5/~m stationary phase DB-5 column. The oven was temperatureprogrammed from 150 to 300°C at 5°C/min and then held at 300°C for 15 rain. The carrier gas was helium, at a flow rate of 2.5 ml/min, and the carrier gas plus make-up gas at a total flow rate of 30 ml/min. G C mass spectrometry (MS) was performed on a Finnigan M A T SSQ 710 G C - M S system. The G C - M S was operated at 70 eV. G C conditions: 30 m x 0.32 m m DB-5 capillary column temperature programmed from 100 to 25WC at 4°C/rain. The carrier gas was helium at 8~b head pressure, linear velocity c. 30 cm/s.

Chemicals and labeled substrate D,L - 2 - [ m e t h y l - 1 4 C ] m e t h y l m a l o n y l - C o A (55.3 mCi/mmol) was purchased from N E N Research

\l

18:1 18:2

r18:0

/

A

LaJ 16:0

L

._

18:0

B

8

C

6 3 4 nC16,

1 2

nC18 nC17 I 14 10 1 5 9 12 3/

! 57

11

16

FIGURE 1. GC traces of the alkanes derived from female housefly fatty acids after reduction to hydrocarbon, (A), total; (B), saturated; (C), methyl branched. Traces A and B were obtained with a flame ionization detector and C is a selective ion monitor scan at m/z 71 obtained from a GC MS analysis. The fatty acids were isolated, methylated and reduced to deuterated alkanes as described in Materials and Methods.

METHYL-BRANCHED FATTY ACIDS AND THEIR BIOSYNTHESIS IN THE HOUSEFLY 57

100-

A c [

80

C - C - C13 - D , 227

40 184

20

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Except as specified in the text, microsomal and soluble proteins were incubated separately with 2 nmol [methyl~4C]methylmalonyl-CoA in a final volume of 250#1 assay buffer with acetyl-CoA, malonyl-CoA, N A D P H and non-radiolabeled methylmalonyl-CoA at the concentrations indicated in the text. The reactions were performed in a shaking water bath at 30°C and stopped by the addition of 125/~1 of 0.5 M K O H in methanol. Reaction mixtures were then saponified at 80°C for 30 min and acidified with HCI. The total lipids were extracted by the procedure of Bligh and Dyer (1959). Each data point is the average and standard error of three replicates. Lipid samples were assayed for radioactivity in 3 ml of Ecolume TM liquid scintillation solution (ICN Biochemicals Inc., Irvine, Calif.). All assays were performed on a Beckman liquid scintillation counter model 1701 at 94~96% efficiency for ~4C.

150

m/z

57 100 155/156

80 112

60

112

40

20 241

100

50

150

was centrifuged at 105,000g for 90min to obtain the microsomal and soluble enzymes. The microsomes were resuspended in an assay buffer (0.05 M potassium phosphate buffer containing 0.05 M KCI, 1 m M EDTA, 1 m M DTE, and 0.5 #g/ml leupeptin, p H 7.2), and repelleted by centrifugation at 105,000g for 90 min. Protein concentrations of the microsomal and soluble fractions were determined using the Bio-Rad protein assay reagent (Bio-Rad Labs, Richmond, Calif.). The purity of the microsomal and soluble preparations were assured by assay with marker enzymes (Juarez et al., 1992).

Assay of FAS activity using radiolabeled methylmalonylCoA

227 .k

805

z~o

m/z

Spectrophotometric FAS assays 57 100

85

C

I,c

183/184

All experiments were performed as described by Gu

--

et al. (1993). Soluble or microsomal protein was added

C4 - C i C11 - D 85

80

40.

183,

to the assay buffer with 200 p M N A D P H . The reaction was initiated by adding a set of CoA substrates including malonyl-CoA, acetyl-CoA and methylmalonyl-CoA, and followed by a spectrophotomeric assay at 340 nm. Concentrations of malonyl-CoA and acetyl-CoA were 60 and 15 p M, respectively. Methylmalonyl-CoA concentrations varied from 0 to 60/~M. The unit of reaction velocity was nmol N A D P H oxidized per min.

20.

oL_ 0

J 50

Jl,l

,J, 150

100

U

2110

RESULTS 250

m/z

FIGURE 2. Mass spectra of components identified as 2-methylpentadecane (A), 7-methylhexadecane(B) and 5-methylhexadecane(C).

containing, l mM EDTA, l m M DTE, and 0.5 #g/ml leupeptin, pH7.2). The homogenate was then centrifuged for 5 min at 4000 g, 10 min at 5100 g, and 20 min at 14,600g to remove all tissue and cells debris, as well as the mitochondrial fraction. The 14,600g supernatant

Methyl-branched fatty acids in the housefly The fatty acid methyl esters from day 4 females were reduced to their corresponding hydrocarbons [Fig. I(A)], the saturated components separated [Fig. I(B)] and the methyl-branched components isolated by inclusion of most of the straight chain components in molecular sieve [Fig. I(C)]. A small amount of the straight chain 16, 17 and 18 carbon alkanes remained in the sample, and are indicated on the selected ion monitor (SIM) trace m/z 71 as nCl6, n C I 7 and nC18 on Fig. 1(C). The methyl-branched hydrocarbons derived from the corresponding fatty acids were

GARY J. BLOMQUIST et al.

806

analyzed by G C - M S and representative mass spectra are presented in Fig. 2. The dimethyl sulfoxide (DMSO) used in the conversion of the alkyl bromide to alkane was washed three times with hexane but still contained small amounts of hydrocarbon contaminants. In the G C - M S analysis, it was possible to differentiate the alkanes derived from insect fatty acids as they contained a deuterium and thus had a molecular weight one unit higher than the unlabeled contaminants, and all the components listed in Table 1 had a mass ion 1 a.m.u. higher than would an unlabeled alkane. Those peaks in Fig. I(C) which do not have a number associated with them either did not yield an interpretable mass spectrum or did not have an ion showing that a deuterium was present, indicating that it was an impurity and not derived from a fatty acid. 4-Methylalkanes and 2-methylalkanes give very similar mass spectra with strong M-43 ions. 4-Methylalkanes also give a M-71/72 peak, but in relatively small molecules, as is the case here, that difference is lost in the ion series 14 units apart from 57 to 141. Thus, we distinguished the 2-methyl- from the 4-methylalkanes by the m/z 70 < 69 for 2-methylalkanes and the m/z 70 > 69 for the 4-methylalkanes. Figure 2(A) presents the mass spectrum of a compound interpreted as 2-methylpentadecane. The M + at 227 and (M-15) + at 212 demonstrate that it contains one deuterium atom. Its derivation from n-2 methylpentadecanoic acid [the n-x nomenclature indicates the position (x) from the methyl end of the fatty acid] and not n-14 methylpentadecanoic acid was deduced from the ion fragment at 183/184, which also contains a deuterium [Fig. 2(A)]. If the deuterium were at the end of the molecule near the methyl-branch (n-14), then the ion fragments at 183/184 would have been at 182/183. The mass spectrum of the component interpreted at 7-methylhexadecane is presented in Fig. 2(B), and is based on the ion fragments at m/z 112, 155/156 and 241. The M + ion at 241 indicates that the molecule

contains a deuterium atom, and the fragment at 155/156 shows that the deuterium is on this fragment, thus demonstrating that the parent fatty acid was n-7 methylhexadecanoic acid and not n-10 hexadecanoic acid. The mass spectrum [Fig. 2(C)] of 5-methylhexadecane was interpreted in a similar manner, and the data show that it was derived from 5-methylhexadecanoic acid. Table 1 presents the diagnostic ion fragments and equivalent chain lengths (ECL) of the methyl-branched alkanes and the fatty acids from which they were derived. In each case, the M + ion contains one deuterium, and indeed, this was used as the criteria to demonstrate that these alkanes were derived from fatty acids and were not hydrocarbon impurities picked up from D M S O during reduction of fatty acids to alkanes. Seventeen monomethyl fatty acids and one dimethyl fatty acid were identified in extracts from day 4 female houseflies. The areas beneath the G C peaks in the analyses of total fatty acid methyl esters corresponding to methylbranched fatty acids were added together. Between 4~6% of the total fatty acids were methyl-branched components, and no component comprised more than 1% of the total fatty acids.

Fatty acid synthase activity with methylmalonyl-CoA The incorporation of [methyl-~4C]methylmalonyl-CoA into fatty acids was assayed with microsomal and soluble preparations from day 1 and day 4 males and females. This was used to monitor the rate of methyl-branched fatty acid production. The results show that both male and female day 4 housefly microsomes contain FAS activity, with higher methylmalonyl-CoA incorporation rates found in the male microsomes [Fig. 3(A)]. The same level of methylmalonyl-CoA incorporation rate was observed in assay mixtures containing day 4 male and female soluble protein [Fig. 3(B)]. For the day 1 females, which do not produce significant amounts of

TABLE 1. Methyl-branched alkanes derived from methyl-branched fatty acids from the housefly, M. domestica Peak number

Hydrocarbon

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

2-methyltetradecane 3-methyltetradecane 2-methylpentadecane 7-methylhexadecane 5-methylhexadecane 4-methylhexadecane 2-methylhexadecane 3-methylhexadecane 3,7-dimethyhexadecane 7,8-methylheptadecane

(l 1) (12) (13) (14) (15) (16) (17)

6-methylheptadecane 5-methylheptadecane 9-methyloctadecane 7-methyloctadecane 6-methyloctadecane 2-methyloctadecane 3-methyloctadecane

Derived from n-2 methyltetradecnoic acid n-3 methyltetradecanoic acid n-2 methylpentadecanoic acid n-7 methylhexadecanoic acid n-5 methylhexadecanoicacid n-4 methylhexadecanoicacid n-2 methylhexadecanoicacid n-3 methylhexadecanoicacid n-3,7 demethylhexadecanoic acid n-7, n-8 methylheptadecanoic acid n-6 methylheptadecanoic acid n-5 methylheptadecanoic acid n-9 methyloctadecanoic acid n-7 methyloctadecanoic acid n-6 methyloctadecanoic acid n-2 methylocatadecanoic acid n-3 methyloctadecanoic acid

ECL

Diagnostic ion fragments~

14.76 169/170,69 > 70 213(M+) 14.80 184(M-29),213(M+) 15.68 183/184,69 < 70 227(M+) 16.45 I12, 155/156 241(M+) 16.51 85, 183/184 241(M+) 16.59 197/198(M-43)241(M+) 16.67 197/198(M-43)69 > 70, 241(M+) 16.69 183, 211/212(M-29) 241(M+) 17.10 126/127, 155/156, 226, 255(M+) 17.37 113, 126/127, 155, 168/169, 255(M +) 17.40 98/99, 183/184, 255(M+) 17.50 85, 196/197, 255(M+) 18.55 140/141,155/156, 269(M+) 18.36 112, 183/184, 269(M+) 18.41 99, 196/197, 269(M+) 18.65 225/226,69 > 70, 269(M+) 18.67 239, 269(M+)

~The ions of some of the fragments are increased by one unit due to the presence of a deuterium.

METHYL-BRANCHED FATTY ACIDS AND THEIR BIOSYNTHESIS IN THE HOUSEFLY

A

DISCUSSION 0.03

O 0.02

O -.=, o

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male

0.00 0

10

20

30

M e t h y l m a l o n y l - C o A (gM)

B o I::

0.03

The methyl-branching groups of the methyl-branched hydrocarbons in the female housefly were shown to be inserted early in the elongation process by both '3CN M R and mass spectral techniques using [it3C]propionate as a precursor (Dillwith et al., 1982). This implied that propionate, as the methylmalonyl-CoA derivative, was incorporated early in the process during fatty acid synthesis, and not towards the end of the process during the fatty acyl-CoA elongation steps. The finding of relatively large amounts of methyl-branched fatty acids, many with the same methyl-branching patterns as are present in the methyl-branched hydrocarbons, lends further support to this hypothesis. These fatty acids are the presumed intermediates of the methylbranched hydrocarbons. The methyl-branched hydrocarbons of the housefly have methyl branches on the 2, 3, 4, 5, 6, 7, 8, 9, 11, 13, and 15 positions and contain

0.02

A 0

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M e t h y l m a l o n y l - C o A (gM) FIGURE 3. Methylmalonyl-CoA incorporation into fatty acids by the microsomal FAS (A) and the soluble FAS (B) from male and female 4 day, post-emergence houseflies. Each reaction mixture contained 200#M NADPH, 60/~M malonyl-CoA, 15#M acetyl-CoA, 50#g microsomal or 25 #g soluble protein, 2 nmol [methyl]4C]methylmalonyl-CoA, and varying methylmalonyl-CoA concentration in a final volume of 250/11. Concentration of methylmalonyl-CoA was varied from 0 to 301tM. Reactions were performed at 30~'C as described in Materials and Methods. The unit (for incorporation on the ordinate) is nmol methylmalonyl-CoA incorporated into fatty acids. The error bars represent the standard error.

methyl-branched hydrocarbon, similar methylmalonylCoA incorporation rates were observed in the microsomal and soluble proteins as were observed in the day 4 females [Fig. 4(A), (B)].

0

0.01

0 0

y 0.00 ,,,, 0

•

1-day

,

,

,

10

20

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M e t h y l m a l o n y l - C o A (gM)

B

0.04

0 0.03.

C ,.=

0.02 •

c ~

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Effect of methylmalonyl-CoA on fatty acid synthase activity As the methylmalonyl-CoA concentration was increased, activity of the soluble and microsomal FAS decreased in both males and females [Fig. 5(A), (B)]. Although the soluble FAS had much higher activity when no methylmalonyl-CoA was present, there was no difference between soluble and microsomal FAS activity when methylmalonyl-CoA reached 60 #M. The IDs0 for soluble FAS from both males and females was between 3.8 and 7.5/~M, while it was 15 # M for the microsomal FAS. Thus, methylmalonyl-CoA had less of an inibitory effect on the microsomal FAS than on the soluble FAS.

"

,,,, 0

-

• i

10

1-day i

20

i

30

M e t h y l m a l o n y l - C o A (gM) FIGURE 4. Methylmalonyl-CoA incorporation into fatty acids by the microsomal FAS (A) and the soluble FAS (B) from female 1 day and 4 day, post-emergence houseflies. Each reaction mixture contained 200#M NADPH, 60#M malonyl-CoA, 15pM acetyl-CoA, 50/~g microsomal or 25/~g soluble protein, 2 nmol [methyl'4C]methylmalonyl-CoA, and varying methylmalonyl-CoA concentration in a final volume of 250/~1. Concentration of methylmalonyl-CoA was varied from 0 to 30#M. Reactions were performed at 30°C as described in Materials and Methods. The unit (for incorporation on the ordinate) is nmol methylmalonyl-CoA incorporated into fatty acids. The error bars represent the standard error.

808

G A R Y J. B L O M Q U I S T et al.

h

0.3 [] female •

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0.2 0.1

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60

Methylmalonyl-CoA (gM) 1.5

"=

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female male

0.5

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40

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Methylmalonyl-CoA (~tM) F I G U R E 5. Effect of methylmalonyl-CoA on activity of the microsomal FAS (A) and the soluble FAS (B). Each reaction mixture contained 200 p M N A D P H , 60 p M malonyl-CoA, 15 p M acetyl-CoA, 5 0 p g microsomal or 2 5 p g soluble protein, and varying methylmalonyl-CoA concentrations in a final volume of 400pl. Concentration of methylmalonyl-CoA was varied from 0 to 60/~ M. Reactions were followed by spectrophotometric assay as described in Materials and Methods. Velocity unit on the ordinate is nmol of N A D P H oxidized per min. The error bars represent the standard error.

3,7-dimethylalkanes. The hydrocarbons have a number of other methyl-branched components whose shorter chain homologues are not found in the methyl-branched fatty acids. The first report of methyl-branched fatty acids with the same methyl-branching positions as those of the methyl-branched hydrocarbons was in the German cockroach, Blattella germanica (Juarez et al., 1992). In the cockroach, an n-3,11-dimethyl fatty acid was identified and 3, I 1-dimethylnonacosane is the precursor to the female sex pheromone, 3,11-dimethylnonacosan2-one (Chase et al., 1992). The finding of methylbranched fatty acids with many of the same branching positions as the hydrocarbons in both the German cockroach (Juarez et al., 1992) and now in the housefly suggest that this may be a general phenomenon in insects, and further work to determine if this is the case is being pursued in our laboratory. Only one dimethyl-branched fatty acid was detected in housefly fatty acids, which may reflect an efficient conversion of multimethyl-branched fatty acid intermediates to hydrocarbon, as the female housefly has abundant

amounts of dimethyl-branched hydrocarbons (Nelson et al., 1981). Relatively large amounts of methyl-branched fatty acids were observed in whole insect extracts, which makes it somewhat surprising that they were not reported in early studies of housefly lipids (Fast, 1970). There are several possible explanations for this. The largest individual methyl-branched component comprises <1% of the total saturated fraction, and if the major component (16:0) were on scale, would be almost undetectable. Furthermore, many of the earlier studies analyzed total fatty acids, and many of the methylbranched fatty acids would be obscured by the relatively large peaks from unsaturated fatty acids. Lastly, most of the earlier studies used packed columns, and they would not have efficiently separated the methyl-branched components as do capillary columns. Three major processes are involved in long chain hydrocarbon biosynthesis. First, FAS synthesizes fatty acids from acetyl-CoA and malonyl-CoA. For methylbranched components, methylmalonyl-CoA is inserted in place of a malonyl group in the early stages of chain synthesis (Dwyer et al., 1981; Dillwith et al., 1982; Blomquist et al., 1987b; Chase et al., 1990). Second, a microsomal fatty acyl-CoA elongase system elongates fatty acids to very long chain moieties (Vaz et al., 1988; Tillman-Wall et al., 1992) which are then converted to the corresponding hydrocarbons by a third system, a reductive decarbonylase (Cheesebrough and Kolattukudy, 1984, 1988; Dennis and Kolattukudy, 1992). FAS has been studied in only a few insect species (Stanley-Samuelson et al., 1988) and it appears similar in structure to vertebrate FAS. A novel microsomal FAS was proposed to be involved in methyl-branched fatty acid biosynthesis of the German cockroach (Juarez et al., 1992; Gu et al., 1993). The fatty acyl-CoA elongase system contains four enzymatic activities and is a microsomal enzyme which has not been purified to homogeneity from any source (Cinti et al., 1992). During the pupal stages of the cabbage looper, Trichoplusia ni, labeled acetate and propionate were efficiently incorporated into methyl-branched lipids at times when the soluble FAS activity was very low or undetectable (de Renobales et al., 1989), indicating that a FAS other than the soluble FAS was involved in producing these lipids. Furthermore, a kinetic study comparing the microsomal and soluble FAS in the German cockroach (Gu et al., 1993) indicated that the microsomal FAS from integument tissue played a key role in methyl-branched fatty acid synthesis by incorporating methylmalonyl-CoA into growing fatty acid chains. The data presented in this paper do not answer the question concerning the relative contribution of the microsomal and soluble FAS in methyl-branched lipid synthesis. The microsomal FAS from the housefly has been purified to homogeneity (P. Gu, W. W. Welch and G. J. Blomquist, unpubl, data), and kinetic studies with purified FAS are underway. Four-day old females produce large amounts of

METHYL-BRANCHED FATTY AC1DS AND THEIR BIOSYNTHESIS 1N THE HOUSEFLY methyl-branched hydrocarbons whereas day 1 males and females and day 4 males produce very small a m o u n t s . T h u s , if the i n c o r p o r a t i o n o f m e t h y l m a l o n y l C o A by F A S r e g u l a t e d m e t h y l - b r a n c h e d lipid p r o d u c t i o n , a d i f f e r e n c e b e t w e e n the a c t i v i t y o f d a y 4 f e m a l e s c o m p a r e d to d a y 1 m a l e s a n d f e m a l e s a n d d a y 1 f e m a l e s w o u l d h a v e b e e n e x p e c t e d . T h e results, in which day 4 males have higher activity than day 4 f e m a l e s s t r o n g l y suggest t h a t m e t h y l - b r a n c h e d h y d r o c a r b o n p r o d u c t i o n is n o t r e g u l a t e d at the level o f F A S . T h e s y n t h e s i s o f ( Z ) - 9 - t r i c o s e n e , the m a j o r sex p h e r o m o n e c o m p o n e n t in the f e m a l e h o u s e f l y ( C a r l s o n et al., 1971), is r e g u l a t e d by the 20-hydroxyecdysone ( B l o m q u i s t et al., 1992) i n d u c e d c h a n g e in the a c y l - C o A e l o n g a t i o n s y s t e m such t h a t 2 4 : 1 - C O A is n o t efficiently e l o n g a t e d in insect p r o d u c i n g sex p h e r o m o n e ( T i l l m a n W a l l et al., 1992). T h e i n c r e a s e d a m o u n t o f 24: l - C o A t h e n p r e s u m a b l y leads to an i n c r e a s e d p r o d u c t i o n o f ( Z ) - 9 - t r i c o s e n e . It is likely t h a t the a c t i v i t y o f the f a t t y a c y l - C o A e l o n g a t i o n s y s t e m also r e g u l a t e s m e t h y l b r a n c h e d h y d r o c a r b o n synthesis, a n d studies are in p r o g r e s s to e x p l o r e this possibility.

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Acknowledgements--This work was supported in part by NSF grant

IBN-9220092. Mass spectral data were acquired on an instrument purchased with NSF grant No. DIR-9102839. This work is a contribution of the Nevada Agricultural Experiment Station. We thank Marilyn Kuenzli for technical assistance and David Quilici for performing the mass spectral analyses.